Bacterial, yeast, plant, insect, and mammalian cell expression systems are currently used to produce recombinant peptides, with varying degrees of success. One goal in creating expression systems for the production of heterologous peptides is to provide broad based, flexible, efficient, economic, and practical platforms and processes that can be utilized in commercial, therapeutic, and vaccine applications. For example, for the production of certain peptides, it would be ideal to have an expression system capable of producing, in an efficient and inexpensive manner, large quantities of final, desirable products in vivo in order to eliminate or reduce downstream reassembly costs.
Currently, bacteria are the most widely used expression system for the production of recombinant peptides because of their potential to produce abundant quantities of recombinant peptides. Bacteria generally do not glycosylate, acetylate, acylate, phosphorylate, or gamma-carboxylate eukaryotic proteins, and, therefore, are limited in their capacities to produce, in vivo, certain types of heterologous eukaryotic peptides that require post-translational modifications. Additional steps modifying the bacterially produced eukaryotic peptides are likely to increase the time and reduce the overall yield of peptide production, diluting many of the advantages of the bacterial host expression system. Therefore, alternative non-bacterial expression systems have been utilized to overcome the inherent limitations of bacterial expression systems.
One particular host expression systems analyzed for their ability to produce heterologous peptides has been whole plants and plant cell suspension cultures. The safe and inexpensive culture of plants provides an improved alternative host for the cost effective production of certain peptides. This alternative is particularly attractive where the protein is complex, requires glycosylation, needs to be free of human or animal infectious viruses, and bacterial toxins.
Whole plants offer the advantages of being relatively inexpensive to grow in vast quantities, with the potential for a great yield of the desired recombinant protein from the large biomass of the harvested transgenic or viral infected plants. As a result, whole plants have been developed as expression systems for commercial production of biopharmaceutical proteins intended for human or veterinary administration.
Significant time and resources have been spent on trying to improve the cost of production and yield of heterologous proteins in non-bacterial systems in order to take advantage of the inherent abilities of these systems. While progress has been made in both of these areas, additional processes and platforms for the production of heterologous peptides in non-bacterial expression systems would be beneficial.
Viruses and Virus Like Particles
One approach for improving peptide production in host cell expression systems is to make use of the properties of infectious recombinant viruses to produce recombinant peptides of interest. The use of infectious viruses in plant host systems is particularly well known. See, for example, Porta & Lomonossoff, (2002) “Viruses as vectors for the expression of foreign sequences in plants,” Biotechnology and Genetic Enginering Reviews 19: 245-291.
Recent strategies have focused on the production of heterologous peptides in virus like particle (VLP) structures. In general, encapsidated viruses include a protein coat or “capsid” that is assembled to contain the viral nucleic acid. Many viruses have capsids that can be “self-assembled” from the individually expressed capsids, both within the cell the capsid is expressed in (“in vivo assembly”), and outside of the cell after isolation and purification (“in vitro assembly”). Ideally, capsids are modified to contain a target recombinant peptide, generating a recombinant viral capsid-peptide fusion. The fusion peptide can then be expressed in a cell, and, ideally, assembled in vivo to form recombinant viral or virus-like particles.
The production of heterologous proteins via virus capsid fusion proteins assembled into VLPs in plants has been met with varying success. See, for example, C Marusic et al., J Virol. 75(18):8434-39 (September 2001) (use of infectious helical potato virus X in whole Nicotiana benthamiana to express virus capsids terminally fused to an antigenic, six amino acid HIV peptide, with in vivo formation of the recombinant virus particles); F R Brennan et al., Vaccine 17(15-16):1846-57 (9 Apr. 1999) (use of infectious cowpea mosaic virus or helical potato virus X capsids terminally fused to an antigenic, Staphylococcus aureus peptide, with in vivo formation of recombinant virus particles expression in whole cowpea plants (Vigna uniquiculata)); C Porta et al., Intervirology 39(1-2):79-84 (1996) (describing an infectious cowpea mosaic virus expressing a chimeric coat protein including an antigenic HIV sequence in whole plants).
U.S. Pat. No. 5,874,087 to Lomonossoff & Johnson describes production of infectious plant viruses, in plant cells or whole plants, wherein the viral capsids are engineered to contain a biologically active peptide, such as a hormone, growth factor, or antigenic peptide. A virus selected from the genera Comovirus, Tombusvirus, Sobemovirus, and Nepovirus is engineered to contain the exogenous peptide encoding sequence and the entire engineered genome of the virus is expressed to produce the recombinant virus. The specification stresses that multiplication of the modified virus is a central part of the invention.
U.S. Pat. No. 6,232,099 to Chapman et al. describes the use of infective, rod-shaped viruses to produce foreign proteins connected to viral capsid subunits in whole plants. Rod-shaped viruses, also classified as helical viruses, such as potato virus X (PVX) have recombinant peptides of interest inserted into the genome of the virus to create recombinant viral capsid-peptide fusions. The recombinant, infective virus is then used to infect a plant cell of a whole plant, wherein, the virus actively replicates in the plant cell and further infects other cells, ultimately infecting the entire host plant. Ultimately, the recombinant viral capsid-peptide fusion is purified from the plant.
Chapman et al. also teaches that a limited insertion size is tolerated by icosahedral viruses. Chapman et al. cite WO 92/18618, which limits the size of the recombinant peptide in an icosahedral virus for expression in a plant host cell to 26 amino acids in length, in supporting his assertion. Chapman et al. theorize that a larger peptide present in the internal insertion site in the capsid of icosahedral viruses may result in disruption of the geometry of the protein and/or its ability to successfully interact with other capsids leading to failure of the chimeric virus to assemble.
U.S. Pat. No. 6,042,832 to Koprowski et al. describes fusion capsid proteins comprising a plant virus capsid protein fused to an antigenic polypeptide. The resultant particles are produced in whole plants.
The utilization of infectious recombinant viruses to produce heterologous proteins in capsid fusion proteins is not, however, without its drawbacks. One particularly troubling aspect is the ability of these viruses to mutate in vivo, resulting in capsid proteins that are essentially wild type revertants without the fused heterologous protein of interest, or mutated, non-desirable recombination capsid fusion protein products. See, or example, Porta & Lomonossoff (1996) “Use of viral replicons for the expression of genes in plants,” Molecular Biotechnology 5:209-221; Dolja et al. (1993) “Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene,” J. Virol. 67(10):5968-5975; Dawson et al. (2001) “Assessment of recombinants that arise from the use of a TMV-based transient expression vector,” Virol. 284(2):182-189 (describing the deletion of the foreign inserted gene in inoculated whole plants). The lack of stability of these viral vectors in whole plants potentially reduces the yield of overall protein product, and may lead to inconsistencies and irregularities in the capsid-fusion product. Such irregularities may be particularly troublesome wherein the integrity of the protein product is essential for a particular desired physio-chemical characteristic in the peptide.
As a result of the inherent instability of infectious recombinant viruses in plants, there is still a need in the field of commercial recombinant protein production for an efficient peptide production system that offers plant-system-type benefits.
In addition, the use of whole plants for the production of recombinant peptides also presents potential problems. For instance, long development times, batch to batch variations in product yield, containment issues, the difficulty of applying good manufacturing practice to the early stages of production, the possibility of contamination with agrochemicals and fertilizers, as well as the impact of pests, disease and variable cultivation conditions due to microclimate and soil differences all result in a potential inconsistent host system for the production of recombinant peptides.
Therefore, it is an object of the present invention to provide a stable and consistent plant cell expression system for the production of virus like particles containing capsid fusion proteins.
It is another object of the present invention to provide plant cells for use as host cells in a stable expression system for the production of virus like particles containing capsid fusion proteins.
It is still another object of the present invention to provide processes for the improved production of virus like particles containing capsid fusion proteins in plant cells, including plant cell suspension cultures.
It is yet another object of the present invention to provide novel constructs and nucleic acids for use in plant cell expression system for the production of virus like particles containing capsid fusion proteins.